Fig 2: Caffeine treatment did not affect amyloid pathology and astrogliosis. Quantification of the Aß plaque load in the cortex (Co), subiculum (Subi), dentate gyrus (DG) and thalamus (Th) of 6-month-old 5xFAD mice using a pan-Aß antibody A and a N-terminal specific antibody B showed that caffeine treatment did not alter Aß plaque deposition in any of the brain regions. Staining with the astrocytic marker GFAP revealed comparable immunoreactivity in all four brain regions in untreated and caffeine-treated 5xFAD mice (n = 10–12 per group) (C). Conforming to the amyloid plaque load analysis, no differences in SDS-soluble Aß1–42 levels were detected in the cortex and hippocampus using ELISA (n = 11–12 per group) (D). Quantification of Aß immunoreactivity in the CA1 region of 6-month-old Tg4-42 mice showed no changes upon caffeine treatment (n = 6 per group). Two-way ANOVA (A-C) or unpaired t tests (D, E). Data are given as means ± SD. Scale bar = 100 µm

Fig 3: ShhN protein expression levels in cultured hippocampal neurons. (A) Immunoblot showing increased ShhN protein level in hippocampal neurons as these neurons matured in culture. d, days in culture. Proteins from HEK cells expressing N-terminal fragment of Shh (ShhN) or full-length Shh (ShhFL) assayed in the same blot as controls. The ShhN antibody detects N-terminal fragment Shh (~19 kD) as well as full-length Shh (~45 kD) from HEK cell lysates, whereas in lysates of cultured neurons, ShhN is the dominant Shh species. psd95, a synaptic marker. Additional blots are shown in Fig. S2A,B. (B) Quantification of A. n=number of independent cultures. (C) Immunoblot showing similar ShhN protein level in neuron-enriched cultures (Enriched) and standard cultures (Standard). Gfap and Aldh1L1 are glial markers, Syp, synaptophysin, neuronal synaptic marker. (D) Quantification of C. n=number of independent cultures. (E,F) Neural activity does not change overall ShhN level. PACAP (10 nM), 2 h; Bicuculline (40 µM), 12 h; ttx, tetrodotoxin (2 µM). (G,H) While treatment of cultured neurons with Shh pathway agonist Purmorphamine (3.6 µM) robustly increases Gli1mRNA level (top, bar graph), the overall level of ShhN protein is not changed (bottom, representative blot) and H. Error bars represent s.e.m. **P<0.01, unpaired t-test. The values of ShhN protein level represent the ShhN band intensities normalized to the actin band intensities.

Fig 4: DBN controls astrocyte scar formation and astrocyte reactivity in vivo.A GFAP+ astrocytes at core lesion sites in WT (upper panels) and Dbn-/- (lower panels) brains, 7 days post stab injury (DPI). Magenta lines indicate the stab injury from needle insertion. Magnifications show stab lesions, with “palisading” astrocytes extending towards injury sites in WT mice. Scale bars: 100 µm. Quantification of palisading astrocytes in stab wounds 7 DPI, bar graph shows mean, individual data points and SEM. n = 4 WT and 3 Dbn-/- animals, Student’s unpaired t test, two-sided, **P = 0.0016 t = 6.205 df = 5). B IBA1 + microglia (magenta) and GFAP+ reactive astrocytes (green) in the periphery of scars 7 DPI, in WT (upper panel) and Dbn-/- brains (lower panel). Scale bars: 10 µm. Quantification of soma sizes of IBA1+ microglia. n = 145 WT & 122 Dbn-/- cells from three mice per condition, graph shows box and whisker plots: box extends from 25th to 75th percentiles, central line = median, whiskers comprise all values from minimum to maximum, F test ***< 0.0001, Unpaired t test with Welch’s correction, two-sided; ***P = 0.000000205637175, t = 5.381 df = 201). C NeuN+ neurons (magenta) and GFAP+ reactive astrocytes (green) in scars of WT (upper panel) and Dbn-/- brains (lower panel) 7 DPI. Scale bars: 10 µm. Quantification shows neurons without nuclear NeuN in scars. n = 3 WT and three Dbn-/- mice, bar graph shows mean, individual data points and SEM, Unpaired t test with Welch’s correction, two-sided; *P = 0.0231, t = 6291 df = 2044). D IHC of WT and DBN-/- brains with 30-day post stab lesions. GFAP labeling (green) detects reactive astrocytes; SOX9 labeling (magenta) detects astrocytes. Scale bars in overview images: 200 µm. Scale bars in close up images: 10 µm. Quantification of cytoplasmic/nuclear SOX9 distribution in astrocytes in lesion sites. Single data points of nuclear SOX9 and corresponding error bars are displayed in magenta, cytoplasmic SOX9, and corresponding error bars in green. n = 4–5 animals per condition. E NeuN+ neurons (magenta) and GFAP+ reactive astrocytes (green) in WT (left), Dbn-/- (center) and Dbnfl/fl:CAMK-Cre+/cre brains (right), 30 DPI. Quantifications show the percentage of animals with GFAP+ astrocytes (left graph) and NeuN+ cells (right graph) at lesion sites 30 days post stab wounding. Bar graph shows mean, individual data points and SEM n = 4 WT, five Dbn-/- animals, six Dbnfl/fl:CAMK-Cre animals, (one-way ANOVA DFn = 2, DFd = 12, F = 8.397; Dunnett’s multiple comparisons test Multiplicity adjusted p value: *P = 0.0112). All images in this figure are confocal stacks. Scale bars: 200 µm. Source data are provided as a Source Data file.

Fig 5: Characterization of HA-tagged a2d1 and a2d3 subunits and protein expression levels before and after lentiviral-induced overexpression. A, Schemes of double HA-tagged a2d1 (left) and a2d3 (right) subunits. Purple represents the localization of the HA tag. B, C, Validation of the a2d1 antibodies in Western blots of either untreated HEK293T cells (control, ctrl) or HEK293T cells expressing the a2d1-HA (A) or a2d3-HA (B) subunit. The HA-tagged a2d proteins were detected using either the anti-a2d antibodies (left) or a highly specific anti-HA antibody that served as positive control (right). Validation of the a2d antibodies in live immunocytochemical stainings of DIV16 hippocampal cultures expressing the HA-tagged a2d subunits and GFP to identify transfected cells. Scale bars, 5 µm. D, Representative images of neuronal cultures at DIV16 stained against the HA tag (live, green), GFAP (magenta), and DAPI (blue) to show a2d1-HA-infected neurons, glial cells, and the total cell number, respectively. Glial cells do not express the a2d1 subunit, thus confirming neuron-specific expression. Scale bar, 50 µm. E, F, Exemplary Western blots showing the endogenous (ctrl) and viral-boosted expression of a2d1 (E) or a2d3 (F) in neurons at DIV16. G, H, Lentiviral infection significantly increases total protein level of the a2d1 (G) and a2d3 (H) subunits. GFAP, glial fibrillary acidic protein, DAPI, 4',6-diamidino-2-phenylindole. *p < 0.05, **p < 0.01. Means and n values are given in Extended Data Figure 2-1.